SPECTRAL CHANGES OF LIGNIN PEROXIDASE DURING REVERSIBLE INACTIVATION

The heme environment of lignin peroxidase (LiP) has been investigated by electronic absorption and electron paramagnetic resonance (EPR) spe ctroscopy. Native LiP was a pentacoordinate, high-spin ferric iron wit h a high-spin absorption band at 634 nm and g values at 5.86 and 2.07 in the EPR spectrum. Upon thermal inactivation, calcium ions were rele ased from the enzyme and the Soret absorption decreased and red-shifte d about 2 nm, the high-spin absorption band at 634 nm disappeared, and a low-spin absorption band appeared at 532 nm. The EPR spectrum and t he temperature dependence of electronic absorption spectra revealed th at the heme iron of the thermally inactivated enzyme was a mixture of high- and low-spin states, which was further supported by the changes in the electronic absorption and EPR spectra when cyanide was added to the thermally inactivated enzyme. Addition of various imidazoles or C N- to thermally inactivated enzyme demonstrated that the low-spin heme iron of inactivated enzyme was hexacoordinate with a distal histidine as its sixth ligand, in contrast to the active enzyme, which was pent acoordinate and high-spin. Upon addition of calcium to recover the the rmally inactivated LiP, the reactivated enzyme had absorptions at 408, 502, and 634 nm and g values at 5.86 and 2.07 in the EPR spectrum, wh ich demonstrated that the heme iron of the reactivated enzyme was agai n high-spin and pentacoordinated.